Difference between revisions of "Davidson Missouri W/Davidson Protocols"

Latest revision as of 01:39, 3 August 2010

A. General Lab Information

1. How to Keep a Lab Notebook
2. Common molecular reagents
3. Standard Assembly
4. BioBrick Ends
5. Compatibility of Plasmids
6. Ethanol Precipitate DNA (short protocol)
7. glycerolstocks How to Make Glycerol Stocks of Bacteria

B. Gel Electrophoresis and Purification

1. Pouring an agarose gel
2. Calculate MWs
3. 1kb MW markers
4. Macherey-Nagel Gel Purification (improved 260/230 ratios)l
5. Qiagen QIAquick Gel Purification
6. Qiagen QIAquick Column Regeneration Protocol
7. ElectroElute Gel Purification

C. Digestion, Ligation, Transformation

1. Digest DNA with restriction enzymes
2. Double Digest Guide
3. Shrimp Alkaline Phosphatase
4. Ligation Protocol
5. Choices for Transformation: Heat Shock vs. Zyppy
6. Heat Shock Transformation OR Short version of Heat Shock
7. Zippy Transformation
8. TSS Competent Cell Transformation

D. Minipreps

1. Choices for Mini-Preps: Promega vs. Zyppy
2. Promega miniprep
3. Zippy Miniprep

E. Making New Parts and PCR

1. Building dsDNA with Oligos
2. Setting up PCR mixtures
3. PCR and Mg²⁺ concentration
4. Making dsDNA Using Primer Dimers
5. Clean and Concentrate DNA (after PCR, before digestion)
6. Colony PCR to Screen for Successful Ligations

F. Expression of Phenotypes

1. Using degradation tags on proteins such as GFP
2. Genomic Insertion Protocol
3. When inducing with IPTG, use 3 µL of stock (0.2 g/mL = 20% w/v) to every 1 mL of LB or other liquid.
4. When inducing with Arabinose, use "2 µL of stock" (10% w/v L-Arabinose) "to every 1 mL" of LB or other liquid.
5. When inducing with 3OC6 (HSL), use a 2000 fold dilution of a 10 mg/mL stock solution. We have dissolved in EtOH which is not the best - degrades with time. Keep this cold.
6. List of auto-inducers and their catalog numbers

G. Computer Tools We Use

1. Optimize your Gel
2. Gene Design (Boeke Lab at JHU)
3. Gene Splitting Web Site
4. PCR Primers w/ BioBricks
5. Promega T_m Calculator
6. Oligator making dsDNA from oligos by Steph and Stephen
7. Lance-olator Oligos for dsDNA assembly
8. Access the GCAT-alog of Davidson and MWSU DNA Freezer Stocks
9. Sequencing at MWG Operon
10. Sequencing at Agencourt Bioscience
11. Sequencing at CUGI
12. Analyzing Sequences with aPe
13. Using Apes (A Plasmid Editor)
14. VeriPart for DNA sequences of Registry Parts
15. The Optimus for optimizing codons